Transposon-sequencing across multiple Mycobacterium abscessus isolates reveals significant functional genomic diversity among strains

Abstract

Mycobacterium abscessus (Mab) is a clinically significant pathogen and a highly genetically diverse species due to its large accessory genome. The functional consequence of this diversity remains unknown mainly because, to date, functional genomic studies in Mab have been primarily performed on reference strains. Given the growing public health threat of Mab infections, understanding the functional genomic differences among Mab clinical isolates can provide more insight into how its genetic diversity influences gene essentiality, clinically relevant phenotypes, and importantly, potential drug targets. To determine the functional genomic diversity among Mab strains, we conducted transposon-sequencing (TnSeq) on 21 genetically diverse clinical isolates, including 15 M. abscessus subsp. abscessus isolates and 6 M. abscessus subsp. massiliense isolates, cataloging all the essential and non-essential genes in each strain. Pan-genome analysis revealed a core set of 3,845 genes and a large accessory genome of 11,507. We identified 259 core essential genes across the 21 clinical isolates and 425 differentially required genes, representing ~10% of the Mab core genome. We also identified genes whose requirements were subspecies, lineage, and isolate-specific. Finally, by correlating TnSeq profiles, we identified 19 previously uncharacterized genetic networks in Mab. Altogether, we find that Mab clinical isolates are not only genetically diverse but functionally diverse as well.

Importance: This study investigates the genetic diversity of Mycobacterium abscessus (Mab), a bacteria known for causing difficult-to-treat infections. Researchers performed transposon-sequencing (TnSeq) on 21 different clinical isolates of Mab to identify essential and non-essential genes in each strain. Through this analysis, they identified core genes required for growth across all strains. Interestingly, they also identified genes whose requirement for growth or "essentiality" were subspecies, lineage, and isolate-specific. This study reveals that Mab's genetic diversity translates into significant functional differences among clinical isolates. Insights from this paper lay essential groundwork for future studies exploring the biological and clinical implications of genetic diversity in Mab clinical isolates. Understanding this diversity could guide targeted therapies and offer new insights into managing infections caused by Mab, a growing public health concern.

Keywords: Mycobacterium abscessus; essential genes; functional diversity; genetic diversity; transposon-sequencing.

Fig 1

Phylogenetic relationship of Mab clinical isolates. (A) Phylogenetic tree of 25 Mab clinical isolates and five reference strains. The tree is generated based on genome-wide SNPs, using Gubbins to filter out genomic regions potentially affected by recombination. Strains with transposon-mutant libraries are marked with an “*.” (B) Global phylogenetic tree of Mab clinical isolates using genomes of 307 clinical isolates from reference (11). Strains in the lab are labeled with an arrow. Red arrows indicate strains that have transposon-mutant libraries.

Fig 2

Mab has a large accessory genome. (A) Flower Pot diagram showing the breakdown of core and accessory genes in lab Mab clinical isolates. The outer leaflets represent the number of accessory genes in each clinical isolate. Strains are arranged counterclockwise based on the degree of similarity to the reference strain by the core genome. (B) (Left) Breakdown of accessory genome content in each clinical isolate. (Right) Percentage of accessory gene content shared by the reference strain. (C) Number of ORFs present in the clinical isolates. (D) Categorization of ORFs in the pan-genome.

Fig 3

Summary of ANOVA results. (A) Heatmap depicting the LFC of insertion counts for the 425 differentially required genes in all isolates. (B) Essentiality category of the differentially essential genes in the ATCC 19977 reference strain.

Fig 4

Principal component analysis of ANOVA hits. (A) PC 1 and 2 of differentially required genes by ANOVA. (B) PC 3 and 4. (C) Phylogenetic tree of Mab clinical isolates shaded with rectangles highlighting clusters. (D) Percentage of variance explained by each of the principal components. (E) Plot depicting the top 10 contributing genes to the variance of PC 1 and 2.

Fig 5

Subspecies and lineage-specific gene requirement. (A) Genes specific to the reference ATCC 19977 strain. (B) Subspecies-specific genes that separate abscessus and massiliense subspecies. (C) (Left) Genes that are differentially required in cluster 1. (Right) Schematized phylogenetic relationship of strains in cluster 1.

Fig 6

Network analysis of differentially required genes. (A) Correlation of transposon insertions between three gene pairs. (B) Cluster 7 depicts 13 genes that are correlated with each other. Genes marked with blue stars are shown in panel A. (C) Network analysis depicting five additional clusters of 19 identified. Cluster 7 is highlighted by a dashed circle.

Fig 7

Genetic requirement differences in carbon metabolism genes among clinical isolates. (A) Schematic of genes in central carbon metabolism in Mab. All heatmaps showcase LFC relative to the reference strain. Genes with significant differences in LFC are highlighted with an “*” indicating a P-adj < 0.05. (B) CFU assay of pckA or sdhB clinical isolate knockdowns.